Much effort has been recently focused on the development of procedures for the targeted delivery of pharmaceutical agents to specific sites in a patient. It is known that drug efficacy can be increased when the appropriate target site is efficiently reached, and drug toxicity can be reduced when the total amount of drug administered is minimized. The interest in drug delivery systems applies both to conventional agents, many of which are relatively simple organic molecules, as well as to more complex agents such as oligopeptides, proteins and nucleic acids, etc. One area of recent interest relates to the use of red blood cells (hereafter erythrocytes and RBCs) to deliver therapeutic dosages of drugs to a target site in a patient. Erythrocytes can be "loaded" with biologically active agents by a process in which the cell membranes of the erythrocytes are lysed and one or more agents is added to the erythrocytes, followed by resealing of the cell membranes. Such "loaded" or "carrier" erythrocytes offer a number of advantages as drug delivery and targeting systems because they are biodegradable, can be maintained in the circulatory system for long periods of time, and can be targeted to selected cells such as, for example, macrophages. However, although the use of erythrocytes as drug delivery systems has been investigated by many, the method has not yet developed to the point where it can be applied routinely in a clinical setting.
Processes for the preparation of a suspension of loaded cells in a physiological solution are disclosed in German Patent No. 23 26 244 and in German published Patent Application Nos. (OS) 23 26 161 and 24 95 119, in which the erythrocyte cell membranes are lysed by either osmotic pressure or an electrical field, respectively.
U.S. Pat. No. 4,224,313, (Zimmermann et al.), discloses a method for preparing a mass of loaded cells suspended in a solution by increasing the permeability of the cell membranes via externally induced osmotic pressure or an electric field, or both. The material to be loaded includes a pharmaceutical agent having a capability, when incorporated in a cell, of prematurely destroying cell membranes, and a stabilizing agent capable of inhibiting the reaction of the pharmaceutical agent with the cell membranes.
U.S. Pat. No. 4,269,826, (Zimmermann et al.), discloses a method for incorporating magnetic substances within loaded cells that can be localized to a specific part of a living body by application of an external magnetic field.
U.S. Pat. No. 4,289,756, (Zimmermann et al.), discloses a method for preferentially accumulating loaded cells within the spleen and liver of a living body.
U.S. Pat. No. 4,478,824, (Franco et al.), discloses a method for incorporating substances into erythrocytes by changing the internal osmotic pressure of RBCs by the action of chemical agents, such as DMSO and glycerol, that can pass through the cell membrane and enter cells by diffusion.
U.S. Pat. No. 4,652,449, (Ropars et al.), discloses a method and apparatus for incorporating materials into erythrocytes using osmotic pressure. The method and apparatus have been used and tested only with large volumes of blood, which limits many applications to the use of homologous blood, i.e., blood pooled from many individual sources. U.S. Pat. No. 4,931,276, (Franco et al.), discloses a method for the encapsulation of non-ionic agents within erythrocytes. The method is of limited effectiveness where the desired agent to be incorporated is not anionic, or is anionic or polyanionic but is not present in the near-isotonic aqueous medium in sufficient concentration to cause needed increases in cell permeability without cell destruction.
Heubsch et al., J. Cell. Physiol., 122:266-272 (1985), demonstrate that cytoskeletal detachment occurs in osmotically swollen cells and that with extreme changes in size, the bilayer membrane is released from a deformational constraint which is present under normal conditions.
Reviews of methods of incorporating substances into cells are presented by Franco et al. in Life Science 32:2763-2768 (1983), Am. J. Hematol. 17:393-400 (1984), and J. Cell. Physiol. 129:221-229 (1986).